Higher-order association states of cellular ERBB3 probed with photo-cross-linkable aptamers.
Nucleic acid aptamers are rapidly gaining prominence as diagnostic tools, targeting reagents, and potential therapeutics. To extend the use of aptamers into the biochemical analysis of protein interactions on the surface of live cells, we converted an enzymatically generated RNA aptamer into a photo-cross-linkable affinity tag through the replacement of all uracils with 4-thiouracil. Specifically, we converted a previously selected, inhibitory aptamer that binds the soluble extracellular domains of the ERBB3 receptor into a targeted and highly specific cross-linking reagent in a live cell setting. Since the photo-cross-linkable aptamer has two functionalities, targeted and highly selective as well as unspecific cross-linking capability, the attachment of this inhibitory aptamer converts ERBB3 into a passive and signaling incompetent probe of its immediate receptor environment. This approach detects receptor clustering of endogenous ERBB3 in the breast cancer cell line MCF7 at levels as low as 25000 receptors per cell and at aptamer concentrations as low as 20 nM. Our analysis also indicates that ERBB3 receptors are apparently segregated from ERBB2 receptors in their resting state, and both ligand-activated ERBB3 and ERBB2 do not share the same microenvironment as inactive ERBB3.
Park
Euisun
E
Department of Biochemistry and Molecular Biology, University of Miami, Miami, Florida 33101-6129, USA.
Baron
Rose
R
Landgraf
Ralf
R
D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D010777:Photochemistry / Q000379:methods; D017434:Protein Structure, Tertiary / Q000502:physiology; D020893:Receptor, ErbB-3 / Q000737:chemistry / Q000378:metabolism*; D013889:Thiouracil / Q000737:chemistry
D052157:Aptamers, Nucleotide; D003432:Cross-Linking Reagents; D013889:Thiouracil; D020893:Receptor, ErbB-3